Mapping gold-labeled receptors on cell surfaces by backscattered electron imaging and digital image analysis: studies of the IgE receptor on mast cells

The response of cells to signaling molecules such as hormones, growth factors, and immune mediators that bind to cell-surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL-2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold-conjugated probes that bind directly or indirectly to the IgE-receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold particles.     

Identification of cells by backscattered electron imaging of silver stained bulk tissues in scanning electron microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanning electron microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Visualization of the cytostome in Trypanosoma cruzi by high resolution field emission scanning electron microscopy using secondary and backscattered electron imaging

High resolution scanning electron microscopy was used to analyze the surface of epimastigote, amastigote and trypomastigote forms of Trypanosoma cruzi. Significant differences were observed between these forms and in different areas of the same cell. The cytostome found in amastigote and epimastigote forms could be easily visualized in images, which resemble those obtained only using the freeze-fracture technique. In contrast to other areas of the cell surface, the region of the cytostome, localized close to the flagellar pocket, showed a rugous surface and an opening with a diameter of 90 nm. Gold-labeled concanavalin A binds to the whole cell surface. However, the extent of binding was much higher in the region of the cytostome. The results obtained show that high resolution scanning electron microscopy is a powerful technique for analyzing the surface of protozoa.     

Mass distributions of coated vesicles isolated from liver and brain: analysis by scanning transmission electron microscopy

Populations of coated vesicles purified from bovine brain (BCV) and from rat liver (LCV) have been characterized with respect to the parameters of mass and diameter by analysis of scanning transmission electron micrographs of unstained specimens. Coated vesicles from both sources are heterogeneous, particularly in their masses. The respective distributions, compiled from mass measurements of many individual particles, are complex and markedly different. BCV range from 20 Mdaltons to approximately 100 Mdaltons with a weighted average of 35 Mdaltons: most BCV (80%) lie between 20 and 40 Mdaltons, including peaks at approximately 26 Mdaltons and at approximately 34 Mdaltons. In contrast, LCV masses tend to be substantially higher, ranging from 20 to 220 Mdaltons with a weighted average of 66 Mdaltons. There is a prominent subpopulation at approximately 35 Mdaltons, and 59% of all LCV belong to a broad peak between 50 and 120 Mdaltons. The Kolmogorov- Smirnov distribution-free test was used to affirm the statistical reproducibility of these isolates. BCV diameters vary from 50 to 90 nm, and those of LCV from 50 to 150 nm. Both protein compositions, determined by SDS PAGE, are dominated by clathrin and they are generally similar, except that corresponding secondary bands, notably the clathrin-associated light chains, appear to have lower molecular weights in the case of LCV. From consideration of the joint mass- diameter distribution, it is apparent that coated vesicles of a given diameter vary considerably in mass and that this variation is due primarily to widely differing amounts of material enclosed within the clathrin coat.     

Glycolipid storage material in Fabry's disease: a study by electron microscopy, freeze-fracture, and digital image analysis

The glycolipid storage material in Fabry's disease was studied by electron microscopy of thin-sectioned (TS) and freeze-fractured (FF) specimens. In the kidney all deposits were found to be located in lysosomes, arranged as lamellar stacks. Deposits in the heart consisted of intracytoplasmic concentric whirls or folded lamellar structures. High resolution TS micrographs disclosed various defects in the lamellar structure. For stabilization, such defects require additional amphiphilic, surface-active molecules. These molecules could interact with other cellular constituents. The lamellar periodicity of the deposits in FF specimens was determined by reconstruction of the three-dimensional fracture face by digital image analysis. Homogeneous multilamellar deposits exhibited a periodicity of 14-15 nm, contrasting with the conventional estimates of 4-5 nm on TS micrographs. This difference is explained by better preservation of the physiologic hydrated state in FF specimens, with 1 vol of lipids binding 2 vol of water. Inhomogeneous structures with an even higher state of hydration included water lenses between the sheets. The strong hydration obviously contributes to the enlargement of the intracellular glycolipid deposits.     

Inter- and intraspecific structural variations among intervascular pit membranes, as revealed by field-emission scanning electron microscopy

The structure of the intervascular pit membranes of four dicotyledonous species (Salix sachalinensis, Betula platyphylla var. japonica, Acer mono, and Fraxinus mandshurica var. japonica) was examined by field-emission scanning electron microscopy. The intervascular pit membranes of F. mandshurica var. japonica had thin surface layers and a dense middle layer, while no similar middle layer was detectable in the other three species. In F. mandshurica var. japonica, the entire area of each pit membrane was densely covered with microfibrils. In the other three species, by contrast, openings were found in the pit membranes. In some of the intervascular pit membranes of S. sachalinensis, B. platyphylla var. japonica, and A. mono, microfibrils were sparsely interwoven in small areas of the pit membranes and openings of up to several hundred nanometers in diameter were present in such regions. These porous regions tended to be located in peripheral areas of pit membranes. In S. sachalinensis and B. platyphylla var. japonica, ethanol-soluble extracts, whose chemical nature and function remain unknown, were heavily distributed over the intervascular pit membranes. Our observations suggest that the structure of intervascular pit membranes is more complicated than has previously been acknowledged.     

Noncytopathic hepatitis A virus induces surface alterations in LLC-MK2 cells revealed by thin sections,negative staining,and scanning electron microscopy

Previous electron microscope studies of ultrastructural events during hepatitis A virus replication in experimentally infected cells have used only ultrathin section techniques. Nevertheless, no important differences were observed between infected and uninfected cells. This study was carried out using scanning electron microscopy and negative staining of whole LLC-MK2 cells grown directly on grids covered with support membranes, and then infected with an hepatitis A virus strain. Thin sections of infected and uninfected controls were also analyzed. An intricate web of projections forming a net between cell interfaces was observed only in infected cells. Some of these projections were more than 700 nm long and had ballooning tips. Nevertheless, HAV particles were not visualized in the infected cells.     

Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.     

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.     

Heterogenous distribution of free calcium and propagation of calcium transient in gastric parietal cells revealed by digital imaging microscopy

Spatial and temporal changes of cytoplasmic free calcium concentration ([Ca2+]i) in single parietal cells of guinea pig were investigated with a digital imaging microscope equipped with a microspectrofluorometer, using a Ca2+-sensitive dye, fura-2. Intracellular distribution of [Ca2+]i was not homogeneous, but there were two kinds of [Ca2+]i gradient in the resting parietal cells, one a continuous gradient increasing towards the plasma membrane and a second discontinuous gradient (Ca2+ plateau) in some restricted regions of the cytoplasm. When treated with gastrin, only about 40% of parietal cells in the gastric gland responded with an almost twofold increase in the average resting [Ca2+]i of 52.4 +/- 7.1 nM. In the responding cells, the discontinuous plateaus transiently enlarged to the entire cytoplasm. In marked contrast, all of these cells responded to Ca2+ ionophore ionomycin. We also found that when provoked by gastrin Ca2+ transient in the parietal cells in the gastric gland often propagated to some adjacent cells, and occasionally spontaneous Ca2+ transient and oscillation were observed even in the resting state.     

External sense receptors in microdrile oligochaetes (Annelida,Clitellata) as revealed by scanning electron microscopy: Typology and patterns of distribution in the main taxonomic groups

This work summarizes the observations on 30 species of microdriles belonging to the families Naididae (Rhyacodrilinae, Pristininae, Naidinae, Phallodrilinae, and Tubificinae), Phreodrilidae, Lumbriculidae, and Enchytraeidae using scanning electron microscopy. The lumbricid Eiseniella tetraedra, a megadrile species common in typical microdrile habitats, was used for comparison. Microdriles display external ciliate sense structures along the entire body; even at the clitellum and in budding and regeneration zones. According to the shape of the cilia, these sense structures can be divided into receptors of blunt cilia, receptors of sharp cilia, and composed receptors. Sense receptors can be morphologically unconspicuous or clearly defined on sensory buds or papillae. All microdriles studied have receptors of blunt cilia. Enchytraeids have characteristic receptors of short cilia. Pristina (Pristininae), Chaetogaster, Ophidonais, and Stylaria (Naidinae) have receptors of long blunt cilia. Composed receptors were found only in some microdriles and E. tetraedra. Receptors of sharp cilia have been found in most microdriles. Enchytraeids might be the only exception, but sharp cilia are probably present in the amphibiotic Cognettia sphagnetorum. Sensory cells with long sharp cilia might play a rheoreceptor role, and their presence in E. tetraedra and C. sphagnetorum would imply the reappearing of an ancient character that was probably lost with the transit from aquatic to terrestrial habitats. Some lumbriculids have ciliated fields. Anatomically, these structures appear as intermediate between the typical isolate sensory structures of microdriles and the sensillae of the hirudineans. The general pattern in microdriles is that uniciliate receptors and multiciliate receptors are separated, which supports the presumed aquatic origin of the clitellates. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Chromosomes and karyotype analysis of a liver fluke, Opisthorchis viverrini, by scanning electron microscopy

Opisthorchis viverrini, a human liver fluke, has been categorized as the carcinogenic organism according to the strong association with carcinogenesis of cholangiocarcinoma (CCA). The infection of this food-borne parasite is a major impact on the health of humans, especially CCA patients in the northeast of Thailand. Taxonomy, morphology, epidemiology and molecular study of O. viverrini have been publicized increasingly but the precise karyotypic study is still incomplete. In this study, the chromosomes of O. viverrini were prepared from the testes of adult worms retrieved from metacercariae infected-hamsters. The chromosomes of O. viverrini were identified in haploid (n=6) meiotic metaphase and in diploid (2n=12) mitotic metaphase by light microscopy. The chromosome number, length and nomenclature of each chromosome were determined by scanning electron microscopy. The six chromosomes consist of one large-sized metacentric, one medium-sized metacentric, two small-sized metacentric, one small-sized submetacentric and one small-sized acrocentric chromosomes with the lengths of 2.84±0.03, 2.12±0.10, 1.71±0.13, 1.44±0.04, 1.23±0.03 and 0.84±0.13 μm, respectively. This is the first karyotype analysis of O. viverrini with defined complete nomenclature.     

Morphological diversity of setae on the grooming legs in Anomala (Decapoda: Reptantia) revealed by scanning electron microscopy

The fifth pereiopods (P5) in Anomala are specialized appendages used mainly for grooming. We studied the articulated cuticular outgrowths, setae, on the distal segments of the P5 in 40 species from 18 Anomala families using light and scanning electron microscopy. Five general classes of setae can be found on the P5: serrate, serrulate and simple setae which all appear bristle-like, and tooth-like and scale-like cuspidate setae. We classified the bristle-like setae according to criteria of shape and the arrangement of distinct outgrowths – denticles and setules – on the shaft of the seta. In this way we were able to distinguish eleven mainly serrate and serrulate types of seta. Some setal types imply homology due to their distinctness and could thus help to solve problematic phylogenetic questions. One setal type, for example, is only present in pagurid hermit crabs and king crabs, which corroborates the theory that these two morphologically very dissimilar groups are, in fact, closely related.     

Structural analysis of human and bovine alpha-fetoprotein by electron microscopy, image processing, and circular dichroism

The images of human and bovine alpha-fetoprotein molecules have been enhanced by combining dark-field electron microscopy with a laser-assisted optical system. This system filters out random background noise while permitting true averaged signal reconstruction of the molecule. A single averaged molecular image was digitized into a matrix, each pixel being assigned a gray scale level to produce a relative mass map for each molecule. These maps were interpreted from the alpha-helix, beta-form, and random coil of the purified proteins as determined by circular dichroism. Results showed that both molecules are "U shaped", apparently monomeric, with outside dimensions of approximately 80 A. Both molecules have asymmetrical structural features, notably three mass dense regions at both extremities and at the vertex of the molecules. Circular dichroism data suggest a high degree of similar stabilized alpha-helix and extensive beta-form in these regions. Mass map analysis of hAFP correlates with the subdomains organized by disulfide bridges.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.